EP3024841B1 - Détection de virus - Google Patents

Détection de virus Download PDF

Info

Publication number
EP3024841B1
EP3024841B1 EP14736939.1A EP14736939A EP3024841B1 EP 3024841 B1 EP3024841 B1 EP 3024841B1 EP 14736939 A EP14736939 A EP 14736939A EP 3024841 B1 EP3024841 B1 EP 3024841B1
Authority
EP
European Patent Office
Prior art keywords
ligand
moiety
nanoparticle
influenza virus
probe
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP14736939.1A
Other languages
German (de)
English (en)
Other versions
EP3024841B8 (fr
EP3024841A1 (fr
Inventor
Rob FIELD
Martin Rejzek
Abdul Rashid
David Andrew Russell
María José MARÍN ALTABA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Iceni Diagnostics Ltd
Original Assignee
University of East Anglia
Plant Bioscience Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of East Anglia, Plant Bioscience Ltd filed Critical University of East Anglia
Publication of EP3024841A1 publication Critical patent/EP3024841A1/fr
Application granted granted Critical
Publication of EP3024841B1 publication Critical patent/EP3024841B1/fr
Publication of EP3024841B8 publication Critical patent/EP3024841B8/fr
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters

Definitions

  • the present invention relates generally to methods and materials for use in the detection of influenza viruses.
  • the influenza virus has two types of surface glycoproteins, haemagglutinin (HA) and neuraminidase (NA).
  • the HA recognizes sialic acids present on the surface of host cells and binds to these carbohydrates in order to infect the cell and the NA releases progeny virus from the infected cell.
  • Measures to prevent a new influenza virus pandemic involve both vaccination and antiviral drugs, the latter ideally administered within 48 h of the infection.
  • influenza The effective use of antivirals requires rapid and early diagnosis.
  • Current methods for the detection of influenza include: molecular identification of influenza isolates including reverse-transcription PCR, immunofluorescence antibody staining, virus isolation in cell culture or in embryonated chicken eggs, and serological diagnosis by haemagglutination inhibition or by microneutralization. [3b] All of these methods are time-consuming, taking several hours or even days for results to be obtained, and also require specialist equipment and trained analysts.
  • Gold nanoparticles (ca. 16 nm in diameter) in aqueous suspension exhibit an intense red color due to their surface plasmon absorption band. This optical property is distance-dependent and upon aggregation of the metal nanoparticles the solution changes color. The color change, readily observed with the naked eye, is due to the coupling interactions between the surface plasmon fields of the particles.
  • Gold nanoparticle-based colorimetric assays have been reported [4] for the detection of a variety of species, including oligonucleotides, metal ions, anions, small organic molecules and proteins, a field reviewed recently by Rotello et a / ..
  • glyconanoparticles By functionalizing metal nanoparticles with specifically synthesized carbohydrate ligands, glyconanoparticles can be created. [6] Glyconanoparticle-based colorimetric assays have been used for for the detection of lectins, calcium ions, and cholera toxin. [7]
  • Gold nanoparticles have been used for the inhibition of influenza virus.
  • Papp et al. employed 14 nm gold nanoparticles functionalized with a sialic-acid-terminated glycerol dendron to inhibit X31 influenza virus (a reassortant H3N2 influenza virus carrying the HA and NA genes of A/Aichi/2/68).
  • gold nanoparticles coated with a phosphonate ester analogue of the influenza therapeutic Oseltamivir, [9] with mercaptoethanesulfonate and mercaptosuccinic acid, [10] and gold nanorods functionalized with ssRNA [11] have also been used for the inhibition of influenza virus.
  • Gold nanoparticles have also been used for the detection of influenza virus.
  • Influenza A/Puerto Rico/8/34 (PR8) (H1N1) virus has been detected using antibody-functionalized gold nanoparticles and dynamic light scattering.
  • Gold nanoparticles functionalized with a chemically unmodified monomer of sialic acid have been used to colorimetrically detect influenza B viruses of the B/Victoria and B/Yamagata lineages through the interaction between the sialic acid and the HA on the virus.
  • US 2008/0194801 relates to a reportedly novel library of compounds comprising a spacer with an attachment element on one terminus and a recognition element on the other terminus.
  • the library of compounds can be attached to a solid support and used in sensors and biosensors.
  • WO 2011/130332 relates to glycan arrays that bind specific target HAs and are reported to detect and distinguish between various sub-types and strains of influenza virus. Methods for using the glycan arrays with assays using nanoparticle amplification technique are also reportedly disclosed.
  • WO2008/123844 relates to a method and system for detecting magnetic nanoparticles include measuring a magneto-optical enhancement of TP the plasmon absorption in the optical response.
  • US 2012/0015344 relates to a particulate composition formed from a conductive polymer bound to magnetic nanoparticles.
  • the particulate composition can be formed into a biologically enhanced, electrically active magnetic (BEAM) nanoparticle composition by further including a binding pair member (e.g., an antibody or a fragment thereof that specifically recognizes a virus strain or a virus surface protein) bound to the conductive polymer of the particulate composition.
  • a binding pair member e.g., an antibody or a fragment thereof that specifically recognizes a virus strain or a virus surface protein
  • glyconanoparticles which include a novel glycoconjugate which presents an influenza-specific sialic acid moieity that can discriminate between influenza virus strains by virtue of its structure and the sialic acid linkage specificity of the corresponding HAs. These glyconanoparticles can be used to specifically and rapidly detect influenza viruses.
  • Preferred glycoconjugates are trivalent, which format provides improved binding and specificity properties, and may better interact with the HA [15] .
  • the inventors have demonstrated that ligands which present a sialic acid ⁇ 2,6 galactose recognition group can be used to specifically detect human influenza virus. In other embodiments sialic acid ⁇ 2,3 galactose sequences may be used to preferentially bind avian influenza virus [14] .
  • the invention therefore provides a nanoparticle probe and a method for detecting influenza virus, as well as a process for preparing a nanoparticle probe, and a kit.
  • a nanoparticle probe comprising a plurality of glycoconjugate ligands, each glycoconjugate ligand (GL) having a plurality of sialic acid containing recognition groups (Y) coupled to the nanoparticle via a multivalent core (X), wherein the multivalent core (X) is a trivalent core, whereby there are 3 recognition groups per ligand, wherein the recognition groups on the bioconjugate specifically bind to the hemagglutinin on the target influenza virus, wherein the probe has at least one further type of ligand bound to the nanoparticle, wherein the further type of ligand is a polyethylene glycol (PEG) which does not bind specifically to an influenza virus, wherein each recognition group terminates with the ⁇ -anomer of a sialic acid moiety, and the sialic acid moiety is bound to a monosaccharide through either:
  • a target influenza virus in a sample which method comprises:
  • the target influenza virus is a human influenza virus
  • the signal generated in (c) is different to the signal generated by an avian influenza virus.
  • other specificities may be provided as described below.
  • “Different” in this context means readily distinguishable under the conditions used in (c) e.g. when compared using equivalent samples under comparable conditions.
  • the signal generated by the non-target influenza virus is less than ⁇ 50%, 40%, 30%, 20%, 10%, 5% of the signal generated by the target virus.
  • “Signal” in this context relates to the changes (shift or intensity) of the surface plasmon absorption band. As shown in the Examples below, preferred nanoparticle probes specific for human influenza virus (tested using X31) target showed negligible changes in the plasmonic signal in the presence of an avian virus.
  • Each glycoconjugate ligand comprises a trivalent core, whereby there are 3 recognition groups (Y) per ligand.
  • each glycoconjugate ligand (GL) may be of the formula: wherein: Y is a sialic-acid containing recognition group;
  • Y terminates with the ⁇ -anomer of a sialic acid moiety, and the sialic acid moiety is bound to a pyranose monosaccharide unit (e.g., galactose) through a 2,6 glycosidic bond.
  • a pyranose monosaccharide unit e.g., galactose
  • the sialic acid moiety and monosaccharide are thio linked.
  • Y may further comprise a spacer moiety 'Z' which connects the sialic acid containing moiety to the core group X.
  • This may comprise, for example, an alkylene or alkenylene group which may include amine, amide, ether, ester or thioester linkages, and optionally be interrupted by one or more heteroatoms and/or rings, including aromatic rings (e.g. benzene, pyridine or 1,2,3-triazole), which rings are optionally substituted.
  • m ⁇ 1 e.g. 3.
  • X is a preferably multivalent core moiety; this spaces group Y from the particle, and spaces the sialic-acid moieties in Y apart to optimise binding; X may comprise groups as defined in 'Z'. It may comprise a multivalent carbon atom ("tripodal core") to which three Y recognition groups are linked (e.g. via X L groups):
  • X L1 , X L2 and X L3 may be, for example, -CH 2 -O-CH 2 -.
  • anomeric centre of the sialic acid moiety or moieties of the Y groups are separated from the single multivalent core carbon atom by 20 to 30 bond lengths (e.g., 22 to 25 bond lengths) or 1.5 to 3 nm (e.g., 2 to 2.5 nm).
  • L is a linking moiety e.g. a thiolinkage; this facilitates attachment of the GL to the nanoparticle.
  • Nanoparticles useful in the practice of the invention include those known in the art for use in other nanoparticle probes and will be made of metal (e.g., gold, silver, platinum, cobalt), semiconductor (e.g., Si, CdSe, CdS, and CdS or CdSe coated with ZnS), core shell particles (e.g., gold coated silver particles), alloy particles (e.g. silver and gold alloy), magnetic (e.g., cobalt), and non-metallic (e.g. silicon) colloidal materials.
  • Core shell particles are described in PCT applications PCT/US01/50825 , and PCT/US02/16382 , as well as in U.S. patent numbers 7,147,687 and 7,238,172 .
  • Other nanoparticles composed of materials e.g. that have an affinity for thiol groups may also be used.
  • the nanoparticle is preferably a gold nanoparticle.
  • the size of the nanoparticles is preferably from about 5 nm to about 150 nm (mean diameter), more preferably from about 5 to about 50 nm, most preferably from about 10 to about 30 nm.
  • the diameter of the nanoparticle may be, for example, 3 nm or greater, 3.5 nm or greater, 4 nm or greater, or 4.5 nm or greater.
  • the diameter of the nanoparticle may be, for example, 10 nm or less, 15 nm or less, 20 nm or less, 30 nm or less, 40 nm or less, 50 nm or less, 75 nm or less, 100 nm or less.
  • the gold nanoparticle may have a diameter in the range of, for example, 3.5 nm to 15 nm, 3.5 to 20 nm, 3.5 to 40 nm, 3.5 nm to 50 nm, 3.5 nm to 75 nm, 3.5 nm to 100 nm.
  • a preferred diameter is c.16nm e.g. 12, 13, 14, 15, 16, 17, 18, 19, or 20 nm.
  • the nanoparticle is a gold nanoparticle of around 16nm diameter.
  • Each nanoparticle probe will generally have multiple glycoconjugate ligands attached, spread over the surface of the nanoparticle.
  • the mean number of GL compounds per nanoparticle may be 5, 10, 25, 50, 100, or 200.
  • the probe has at least one further type of ligand bound to the nanoparticle, in addition to the glycoconjugate ligands, wherein the further type of ligand is one which does not bind specifically to influenza virus, and which may be used to modulate density of the glycoconjugate ligand on the surface of the nanoparticle to optimise binding and specificity.
  • the probe comprises polyethylene glycol (PEG), e.g. a thiolated PEG, bound to the solid support.
  • the polyethylene glycol is H(OCH 2 ) 4 S-, bound to the nanoparticle through the sulphur atom.
  • the probe comprises a phase transfer reagent bound to the solid support.
  • the phase transfer reagent is tetraoctylammonium halide, e.g., tetraoctylammonium bromide (TOAB)
  • TOAB tetraoctylammonium bromide
  • the molar ratio of glycoconjugate ligand: further ligand is between 10:90 and 90:10.
  • the further ligand is in excess.
  • Example ratios as disclosed herein are 1:99; 5:95; 10:90; 25:75; 50:50; 75:25: or 90:10.
  • the ratio of glycoconjugate ligand: further ligand is between 15:85 and 35:65 e.g. between 20:80 and 30:70 e.g. about 25:75.
  • the detectable signal is a color change, which may be observable with the naked eye.
  • step (b) causes aggregation of the nanoparticles, wherein said aggregation generates or contributes to the detectable plasmonic signal.
  • the detectable signal is generated within 60, 50, 40, 30, 20, 10 or 5 mins.
  • the methods of the present invention can be used with little or no sample preparation. They can be used, for example, for detecting influenza viruses and/or virus particles in samples of animals and/or humans like swabs, faeces and blood, in environmental samples. In one embodiment, the methods of the present invention are used for detecting influenza viruses and/or virus particles in sputum or saliva samples of animals and/or humans.
  • the nanoparticle probes are utilised in an aqueous suspension.
  • a virus-detection solution comprising an aqueous suspension of a glycoconjugate described above, bound to nanoparticles.
  • concentration of glycoconjugate-covered nanoparticles in the virus-detection solution may be 0.05 nM to 10 nM, for example, 0.1 nM to 7.0 nM, or 1 nM to 5 nM.
  • a sample of interest may be added directly (neat) to the virus-detection solution.
  • a sample of interest may be diluted prior to being added to the virus-detection solution.
  • a detectable plasmonic signal e.g. color change
  • a detectable plasmonic signal e.g. color change
  • Such an assessment with the naked eye can be made more readily against a background of a contrasting color.
  • the observation of a color change is facilitated by spotting a sample of the hybridization solution on a solid white surface (such as silica or alumina TLC plates, filter paper, cellulose nitrate membranes, and nylon membranes, preferably a nylon membrane) and allowing the spot to dry.
  • a solid white surface such as silica or alumina TLC plates, filter paper, cellulose nitrate membranes, and nylon membranes, preferably a nylon membrane
  • the color change may be quantitated by recording the plate image with an optical scanning device such as a flatbed scanner or CCD camera, and analyzing the amount and type of color of each individual spot.
  • an optical scanning device such as a flatbed scanner or CCD camera
  • a color filter e.g. red filter
  • Suitable substrates include transparent solid surfaces (e.g., glass, quartz, plastics and other polymers), opaque solid surface (e.g., white solid surfaces, such as TLC silica plates, filter paper, glass fiber filters, cellulose nitrate membranes, nylon membranes), and conducting solid surfaces (e.g., indium-tin-oxide (ITO)).
  • transparent solid surfaces e.g., glass, quartz, plastics and other polymers
  • opaque solid surface e.g., white solid surfaces, such as TLC silica plates, filter paper, glass fiber filters, cellulose nitrate membranes, nylon membranes
  • conducting solid surfaces e.g., indium-tin-oxide (ITO)
  • the substrate can be any shape or thickness, but generally will be flat and thin.
  • transparent substrates such as glass (e.g., glass slides) or plastics (e.g., wells of microtiter plates).
  • samples may be subject to confirmatory tests like reverse transcription polymerase chain reaction.
  • the glycoconjugate ligands can be attached to different (non-nanoparticle) solid supports.
  • solid support refers to a material having a rigid or semi-rigid surface which a compound as described herein can bond to. Such supports will preferably take the form of small beads, pins/crowns, laminar surfaces, pellets, disks.
  • the solid support may be part of detection system e.g. based on surface plasmon resonance, or surface enhanced Raman spectroscopy.
  • the glycoconjugate ligands can be attached to a fluorescent nanoparticle, e.g., a quantum dot, a fluorescently-tagged polymer bead, a fluorescently-tagged silica bead (e.g., silica nanoparticle) or a fluorescently-tagged magnetic bead (e.g., magnetic nanoparticle).
  • a fluorescent nanoparticle e.g., a quantum dot
  • a fluorescently-tagged polymer bead e.g., a fluorescently-tagged silica bead (e.g., silica nanoparticle) or a fluorescently-tagged magnetic bead (e.g., magnetic nanoparticle).
  • nanoparticle probes as described herein comprise:
  • the recognition group (Y) on the bioconjugate specifically binds to the hemagglutinin on a target influenza virus, which specific binding generates a detectable plasmonic signal which is specific to the influenza virus.
  • the nanoparticle probes comprise a glycoconjugate ligand (GL) attached to a nanoparticle, wherein each GL compound comprises 1 or more, preferably 2, most preferably 3 sialic-acid containing recognition groups (Y), as shown in of formula (I):
  • the GL may be of the formula (II): wherein:
  • m is 3 to 6.
  • m is 3 to 5.
  • m is 3 or 4.
  • m is 3.
  • the sialic-acid containing recognition group, Y comprises a sialic acid moiety attached to one or more monosaccharide units, with the monosaccharide units being attached to the core, X, by a bond or by a spacer moiety.
  • Y terminates with the ⁇ -anomer of a sialic acid moiety, and the sialic acid moiety is bound to a pyranose monosaccharide unit (e.g., galactose) through a 2,3 glycosidic bond.
  • a pyranose monosaccharide unit e.g., galactose
  • Y terminates with the ⁇ -anomer of a sialic acid moiety, and the sialic acid moiety is bound to a pyranose monosaccharide unit (e.g., galactose) through a 2,6 glycosidic bond.
  • a pyranose monosaccharide unit e.g., galactose
  • the anomeric centre of the sialic acid moiety is separated from the multivalent centre of the core moiety X by 20 to 30 bond lengths (e.g., 22 to 25 bond lengths). This is described in more detail hereinafter.
  • the anomeric centre of the sialic acid moiety is separated from the core moiety X by 1.5 to 3 nm (e.g., 2 to 2.5 nm).
  • each sialic-acid containing recognition group Y is independently a moiety of the following formula: wherein:
  • Y has a relative molecular weight of less than 5,000.
  • Y has a relative molecular weight of less than 3,000.
  • Y has a relative molecular weight of less than 2,000.
  • Y has a relative molecular weight of less than 1,000.
  • each core moiety, X is attached to more than one type of sialic-acid containing recognition group, Y (e.g., sialic-acid containing recognition groups with different Sia, Sac 1 , Sac N , Z and or n).
  • Y e.g., sialic-acid containing recognition groups with different Sia, Sac 1 , Sac N , Z and or n.
  • the GL compound may have two different types of sialic-acid containing recognition group: Y 1 and Y 2 , or three different types of sialic-acid containing recognition group: Y 1 , Y 2 and Y 3 .
  • each core moiety, X is attached to only one type of sialic-acid containing recognition group, Y (i.e., all recognition groups have the same Sia, Sac 1 , Sac N , Z and n).
  • sialic acid moiety is a moiety which is related to neuraminic acid, which has the following formula:
  • sialic acid moiety is a moiety of the following formula: or a salt thereof; wherein:
  • C 1-6 alkyl as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a saturated hydrocarbon compound having from 1 to 6 carbon atoms. For groups with more than 3 carbon atoms, the group can be linear or branched.
  • the sialic acid moiety is a moiety of the following formula: or a salt thereof; wherein:
  • Examples of suitable moieties of formulae (V) and (VI) include, for example, the moieties shown in Table I.
  • Table I Examples of sialic acid moieties falling within formulae (V) and (VI) Name Abbreviation Formula Formula Reference N -acetylneuraminic acid Neu5Ac (Sia-1) N- glycolylneuraminic acid Neu5Gc (Sia-2) N -acetyl-9- O- acetylneuraminic acid 9- O -acetyl Neu5Ac (Sia-3)
  • Sia is a moiety of formula (Sia-1), (Sia-2), or (Sia-3).
  • Sia is a moiety of formula (Sia-1) or (Sia-2).
  • Sia is a moiety of formula (Sia-1).
  • Sia moiety of formula (V) above has at least six chiral centres, specifically, the carbon atoms marked with an asterisk (*) in the following formula. Each of the carbon atoms at these positions may be in either ( R ) or ( S ) configuration. Unless otherwise stated, a reference to one enantiomer/diastereomer is intended to be a reference to both enantiomers/all diasteromers.
  • sialic acid moiety is of the following formula:
  • the R C2 group e.g., COOR 1
  • the R C2 group is in the axial position.
  • the R C2 group e.g., COOR 1
  • the R C2 group is in the equatorial position.
  • the sialic acid moiety Sia is the ⁇ -anomer.
  • the sialic acid moiety Sia is the ⁇ -anomer of a compound of formula (VI); that is, a moiety of formula (IX),
  • sialic moieties falling within formula (IX) include, for example, the moieties shown in Table II.
  • Table II - Examples of sialic acid moieties falling within formula (IX) Name Abbreviation Formula Formula Reference N -acetylneuraminic acid Neu5Ac (Sia-1a) N -glycolylneuraminic acid Neu5Gc (Sia-2a) N -acetyl-9- O- acetylneuraminic acid 9- O -acetyl Neu5Ac (Sia-3a)
  • Sia is a moiety of formula (Sia-1a), (Sia-2a), or (Sia-3a).
  • Sia is a moiety of formula (Sia-1a) or (Sia-2a).
  • Sia is a moiety of formula (Sia-1a).
  • Sia is a moiety of the following formula
  • Sia is a moiety of the following formula:
  • W is S.
  • W is C.
  • R C4 is OR 4 .
  • R C4 is NR N4 R N5 .
  • R 1 is H.
  • R 1 is R A1 .
  • R 4 is H.
  • R 4 is R A4 .
  • R 7 is H.
  • R 7 is R A7 .
  • R 8 is H.
  • R 8 is R A8 .
  • R 9 is H.
  • R 9 is R A9 .
  • R N4 is H.
  • R N4 is C 1-6 alkyl.
  • R N4 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R N4 is methyl, ethyl, i -propyl, or n -propyl.
  • R N4 is methyl or ethyl.
  • R N4 is methyl
  • R N5 is H.
  • R N5 is C 1-6 alkyl.
  • R N5 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R N5 is methyl, ethyl, i -propyl, or n -propyl.
  • R N5 is methyl or ethyl.
  • R N5 is methyl
  • R A1 is C 1-6 alkyl.
  • R A1 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R A1 is methyl, ethyl, i -propyl, or n -propyl.
  • R A1 is methyl or ethyl.
  • R A1 is methyl
  • R A4 is C 1-6 alkyl.
  • R A4 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R A4 is methyl, ethyl, i -propyl, or n -propyl.
  • R A4 is methyl or ethyl.
  • R A4 is methyl
  • R A7 is C 1-6 alkyl.
  • R A7 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R A7 is methyl, ethyl, i -propyl, or n -propyl.
  • R A7 is methyl or ethyl.
  • R A7 is methyl
  • R A8 is C 1-6 alkyl.
  • R A8 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R A8 is methyl, ethyl, i -propyl, or n -propyl.
  • R A8 is methyl or ethyl.
  • R A8 is methyl
  • R A9 is C 1-6 alkyl.
  • R A9 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R A9 is methyl, ethyl, i -propyl, or n -propyl.
  • R A9 is methyl or ethyl.
  • R A9 is methyl
  • R P1 is C 1-6 alkyl.
  • R P1 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R P1 is methyl, ethyl, i -propyl, or n -propyl.
  • R P1 is methyl or ethyl.
  • R P1 is methyl
  • R P2 is C 1-6 alkyl.
  • R P2 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R P2 is methyl, ethyl, i -propyl, or n -propyl.
  • R P2 is methyl or ethyl.
  • R P2 is methyl
  • R S1 is C 1-6 alkyl.
  • R S1 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R S1 is methyl, ethyl, i -propyl, or n -propyl.
  • R S1 is methyl or ethyl.
  • R S1 is methyl
  • R N1 is H.
  • R N1 is C 1-6 alkyl.
  • R N1 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R N1 is methyl, ethyl, i -propyl, or n -propyl.
  • R N1 is methyl or ethyl.
  • R N1 is methyl
  • R N2 is H.
  • R N2 is C 1-6 alkyl.
  • R N2 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R N2 is methyl, ethyl, i -propyl, or n -propyl.
  • R N2 is methyl or ethyl.
  • R N2 is methyl
  • R NN2 is C 1-6 alkyl.
  • R NN2 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R NN2 is methyl, ethyl, i -propyl, or n -propyl.
  • R NN2 is methyl or ethyl.
  • R NN2 is methyl
  • R NN2 is CH 2 OR NNN2 .
  • R NNN2 is H.
  • R NNN2 is R NNNN2 .
  • R NNNN2 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R NNNN2 is methyl, ethyl, i -propyl, or n -propyl.
  • R NNNN2 is methyl or ethyl.
  • R NNNN2 is methyl
  • the sialic acid moiety, Sia is attached to a monosaccharide unit, Sac 1 , which is a pyranose-based monosaccharide.
  • the monosaccharide unit Sac 1 is a compound of the following formula: wherein;
  • this monosaccharide unit can exist in two anomeric forms, designated alpha ( ⁇ ) and beta ( ⁇ ).
  • alpha anomeric form the R Q1 attached to anomeric carbon C-1 is cis to the group attached to C-5.
  • beta anomeric form R Q1 attached to anomeric carbon C-1 is trans to the group attached to C-5.
  • Sac 1 moiety of formula (XIII) above has at least five chiral centres, specifically, the carbon atoms at positions 1, 2, 3, 4, and 5, which are each marked with an asterisk (*) in the following formula.
  • Each of the carbon atoms at these positions may be in either ( R ) or ( S ) configuration.
  • a reference to one enantiomer/diastereomer is intended to be a reference to both enantiomers/all diasteromers.
  • Suitable monosaccharide units include pyranose forms of, for example:
  • Sac 1 is a D-galactose unit.
  • Sac 1 has the following formula:
  • Sac 1 is the ⁇ -anomoer of a galactose unit.
  • the monosaccharide unit Sac 1 is bonded to group W of the sialic acid moiety through a glycosidic bond.
  • W is S
  • W this corresponds to an S-glycosidic bond.
  • W is C
  • any of groups R Q1 , R Q2 , R Q3 , R Q4 , and R Q6 can be a bond to group W of the sialic acid moiety, as shown in Table III.
  • R Q1 is a bond to W of the sialic acid moiety.
  • R Q1 is R'.
  • R Q1 is OR'.
  • R Q1 is OR QQ1 .
  • R Q2 is a bond to W of the sialic acid moiety.
  • R Q2 is R'.
  • R Q2 is OR'.
  • R Q2 is OR QQ2 .
  • R Q3 is a bond to W of the sialic acid moiety.
  • Sac 1 is a D-galactose unit linked to Sia through a 2,3 bond.
  • Sac 1 has the following formula: where indicates the point of attachment to W of Sia.
  • Sac 1 is a D-galactose unit linked to Sia through an ⁇ -2,3 bond.
  • Sia is an ⁇ -anomer, and is bonded to a D-galactose unit through a 2,3 glycosidic bond.
  • Sia-Sac 1 are of the following formula:
  • the D-galactose unit is the ⁇ -anomer.
  • R Q3 is R'.
  • R Q3 is OR'.
  • R Q3 is OR QQ3 .
  • R Q4 is a bond to W of the sialic acid moiety.
  • R Q4 is R'.
  • R Q4 is OR'.
  • R Q4 is OR QQ4 .
  • R Q6 is a bond to W of the sialic acid moiety.
  • Sac 1 is a D-galactose unit linked to Sia through a 2,6 bond.
  • Sac 1 has the following formula: where indictates the point of attachment to W of Sia.
  • Sac 1 is a D-galactose unit linked to Sia through an ⁇ -2,6 bond.
  • Sia is an ⁇ -anomer, and is bonded to a D-galactose unit through a 2,6 glycosidic bond.
  • Sia-Sac 1 are of the following formula:
  • the D-galactose unit is the ⁇ -anomer.
  • R Q6 is R'.
  • R Q6 is OR'.
  • R Q6 is OR QQ6 .
  • R QQ1 is H.
  • R QQ1 is R QQQ1 .
  • R QQ2 is H.
  • R QQ2 is R QQQ2 .
  • R QQ3 is H.
  • R QQ3 is R QQQ3 .
  • R QQ4 is H.
  • R QQ4 is R QQQ4 .
  • R QQ6 is H.
  • R QQ6 is R QQQ6 .
  • R QQQ1 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R QQQ1 is methyl, ethyl, i -propyl, or n -propyl.
  • R QQQ1 is methyl or ethyl.
  • R QQQ1 is methyl
  • R QQQ2 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R QQQ2 is methyl, ethyl, i -propyl, or n -propyl.
  • R QQQ2 is methyl or ethyl.
  • R QQQ2 is methyl
  • R QQQ3 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R QQQ3 is methyl, ethyl, i -propyl, or n -propyl.
  • R QQQ3 is methyl or ethyl.
  • R QQQ3 is methyl
  • R QQQ4 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R QQQ4 is methyl, ethyl, i -propyl, or n -propyl.
  • R QQQ4 is methyl or ethyl.
  • R QQQ4 is methyl
  • R QQQ6 is methyl, ethyl, i -propyl, n -propyl, t -butyl, i -butyl, s -butyl or n- butyl.
  • R QQQ6 is methyl, ethyl, i -propyl, or n -propyl.
  • R QQQ6 is methyl or ethyl.
  • R QQQ6 is methyl
  • the compound may include additional monosaccharide units, Sac N , in addition to the sialic-acid-attached monosaccharide unit, Sac 1 .
  • the number of Sac N units present is determined by the integer n.
  • n is 0 to 10.
  • n is 0 to 8.
  • n is 0 to 6.
  • n is 0 to 5.
  • n is 0 to 4.
  • n is 0 to 3.
  • n is 0 to 2.
  • n is 0 or 1.
  • n is 0 (i.e., no Sac N are present).
  • n ⁇ 1 i.e., one or more Sac N are present.
  • n may be 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • n is 1 (i.e., one Sac N unit is present). In such an embodiment, Sac 1 and Sac N form a disaccharide.
  • Suitable monosaccharide units include pyranose forms of, for example:
  • Sac N is selected from one of the monosaccharide units shown in Table IV: Table IV - Suitable Sac N units Name Abbreviation Formula Formula Reference D-galactose Gal (Sac-1) ⁇ -D-galactose ⁇ -Gal (Sac-1a) ⁇ -D-galactose ⁇ -Gal (Sac-1b) D-glucose Glc (Sac-2) ⁇ -D-glucose ⁇ -Glc (Sac-2a) ⁇ -D-glucose ⁇ -Glc (Sac-2b)
  • Sac 1 is bound to a monosaccharide unit of formula (Sac-2b).
  • Z is a single bond.
  • Z is a spacer moiety.
  • the spacer moiety allows the length, composition and rigidity of the sialic-acid containing recognition group, Y to be modified. This permits the sialic-acid containing recognition groups to be presented to the binding sites of haemagglutinin whilst minimising strain in the glycoconjugate ligand compound.
  • Z is a C 1-20 alkylene group (e.g., a C 1-15 or C 1-10 alkylene group).
  • C 1-20 alkylene refers to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a saturated hydrocarbon compound having from 1 to 20 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic.
  • the alkylene group is a linear aliphatic alkylene group.
  • Z is a C 1-20 alkenylene group having 1 to 3 carbon-carbon double bonds (e.g., a C 1-15 or C 1-10 alkenylene group).
  • C 1-20 alkenylene having 1 to 3 carbon-carbon double bonds pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 1 to 20 carbon atoms (unless otherwise specified) and one to three carbon-carbon double bonds, which may be aliphatic or alicyclic.
  • Z is a C 1-20 alkynylene group having 1 to 3 carbon-carbon triple bonds (e.g., a C 1-15 or C 1-10 alkynylene group).
  • C 1-20 alkynylene having 1 to 3 carbon-carbon double bonds pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 1 to 20 carbon atoms (unless otherwise specified) and one to three carbon-carbon triple bonds, which may be aliphatic or alicyclic.
  • the Z groups may optionally include one or more amine, amide, ether, ester or thioester linkages.
  • the Z groups above may be optionally interrupted by one or more heteroatoms and/or aromatic rings (e.g. benzene, pyridine, or 1,2,3-triazole) which rings are optionally substituted.
  • aromatic rings e.g. benzene, pyridine, or 1,2,3-triazole
  • Z comprises an alkylene or alkenylene group which optionally includes one or more amine, amide, ether, ester or thioester linkages, and optionally is interrupted by one or more heteroatoms (e.g., O, S, N) and/or aromatic rings (e.g., pyridine, benzene, 1,2,3-triazole).
  • heteroatoms e.g., O, S, N
  • aromatic rings e.g., pyridine, benzene, 1,2,3-triazole
  • Z comprises a triazole group.
  • Z comprises an amide linkage
  • Z is a C 1-20 amine group (e.g., a C 1-15 or C 1-10 amine group).
  • Z is a C 1-20 amide group (e.g., a C 1-15 or C 1-10 amide group).
  • Z is a C 1-20 ether group (e.g., a C 1-15 or C 1-10 ether group, or a C 1-20 , C 1-15 or C 1-10 polyether group).
  • Z is a C 1-20 ester group (e.g., a C 1-15 or C 1-10 ester group).
  • Z is a C 1-20 thioether group (e.g., a C 1-15 or C 1-10 thioether group, or a C 1-20 , C 1-15 or C 1-10 polythioether group).
  • Z includes a first functional group, A 1 , which connects Z to X.
  • a 1 is selected from:
  • a 1 is a C 6-10 arylene group or a C 5-10 heteroarylene group.
  • a 1 is a C 5-10 heteroarylene group.
  • a 1 is a group obtained by CLICK chemistry.
  • a 1 is a group obtained by 1,3-dipolar cycloaddition.
  • a 1 is a 1,2,3-triazole group.
  • a 1 may be a group having the following formula: wherein the wavy line indicates the point of attachment to the rest of Z, and the asterisk * indicates the carbon to which X is attached.
  • Such a group can be obtained through Huisgen cycloaddition between an azide group and an alkyne group.
  • Z includes a second functional group, A 2 , which connects Z to Sac 1 (when n is 0) or Sac N (when n>0).
  • a 2 is selected from
  • Z is a moiety having the following formula: wherein:
  • d is 3 and e is 5.
  • the core moiety X is a group to which the sialic-acid containing recognition groups, Y, are attached.
  • X is a C 1-30 group to which Y is attached (e.g., a C 1-20 group, or a C 1-15 group).
  • X is a C 1-30 alkylene group (e.g., a C 1-20 alkylene group or a C 1-15 alkylene group).
  • X is a C 1-30 alkenylene group, the group having 1 to 3 carbon-carbon double bonds (e.g., a C 1-20 alkenylene group or a C 1-15 alkenylene group).
  • X is a C 1-30 alkynylene group, the group having 1 to 3 carbon-carbon triple bonds (e.g., a C 1-20 alkynylene group or a C 1-15 alkynylene group).
  • X is a C 1-30 amine group (e.g., a C 1-20 or C 1-15 amine group).
  • X is a C 1-30 amide group (e.g., a C 1-20 or C 1-15 amide group).
  • X is a C 1-30 ether group (e.g., a C 1-20 or C 1-15 ether group, or a C 1-30 , C 1-20 or C 1-15 polyether group).
  • X is a C 1-30 ester group (e.g., a C 1-12 or C 1-15 ester group).
  • X is a C 1-30 thioether group (e.g., a C 1-20 or C 1-15 thioether group, or a C 1-30 , C 1-20 or C 1-15 polythioether group).
  • heteroatoms e.g, O, S, N
  • aromatic rings e.g., benzene, pyridine or 1,2,3-triazole
  • three Y recognition groups are linked to a single carbon atom on the core moiety, as shown in the following formula: wherein: X L1 , X L2 and X L3 are linking groups, and the wavy line indicates the point of attachment to the rest of X.
  • X L1 , X L2 and X L3 are, R X1 OR X2 , R X1 NHR X2 or R X1 SR X2 , where R X1 is a bond or C 1-6 alkylene and R X2 is a bond or C 1-6 alkylene.
  • three Y recognition groups are linked (e.g. via X L groups) to the same single multivalent carbon atom ("tripodal core") of the core moiety, and the anomeric centre of the sialic acid moiety is separated from said single carbon atom by 20 to 30 bond lengths (e.g., 22 to 25 bond lengths).
  • three Y recognition groups are linked to the single carbon atom on the core moiety, and the anomeric centre of the sialic acid moiety is separated from said single carbon atom by 1.5 to 3 nm (e.g., 2 to 2.5 nm).
  • X is a moiety having the following formula: wherein:
  • f is 4 and g is 3.
  • X L1 , X L2 and X L3 are -CH 2 -O-CH 2 - or -CH 2 -S-CH 2 -.
  • X is a moiety of the following formula:
  • the linking moiety L comprises a functional group which attaches the core moiety X to the nanoparticle.
  • L is a sulphur atom, S, which binds to the nanoparticle, i.e., a moiety of the following formula: wherein the wavy line indicates the point of attachment to X and the asterisk * indicates the point of attached to nanoparticle O.
  • L is derived from a 1,2-dithiolane.
  • L is a moiety of the following formula: wherein the wavy line indicates the point of attachment to X and the asterisk * indicates the point of attached to nanoparticle O.
  • L may be derived from the 1,2-dithiolane group of thioctic acid (lipoic acid) or thioctic acid amide, or derivatives thereof.
  • the conjugate is of formula (I): wherein:
  • O in formula (I) is a gold nanoparticle.
  • n 0.
  • d is 3 and e is 5.
  • f is 5 and g is 4.
  • X L1 , X L2 and X L3 are - CH 2 -O-CH 2 -.
  • the conjugate additionally comprises polyethylene glycol, preferably at an HB to PEG ratio of 25:75.
  • Y has the following formula: where the asterisk (*) indicates the carbon atom to which X is attached.
  • the conjugate has the following formula:
  • glycoconjugate ligand compounds which can be attached to a nanoparticle to make nanoparticle probes, and precursors of such compounds.
  • glycoconjugate ligand compound of the following formula: wherein:
  • the linking moiety L* comprises a functional group which can attach the core moiety X to a nanoparticle.
  • L* may be a thiol group (e.g., SH).
  • a reference to carboxylic acid also includes the anionic (carboxylate) form (-COO - ), a salt or solvate thereof, as well as conventional protected forms.
  • a reference to an amino group includes the protonated form (-N + HR a R b ), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group.
  • a reference to a hydroxyl group also includes the anionic form (-O-), a salt or solvate thereof, as well as conventional protected forms.
  • nanoparticle probes described herein form one aspect of the present invention, as do kits comprising them.
  • Glycoconjugates as described herein may be prepared using conventional methods known in the art, or by adapting conventional methods known in the art in conventional ways.
  • Synthesis of sialic acid moieties where R C4 is -NR N4 R N5 can be made using, for example, the methods described in Vonitzstein [33] .
  • the method of making a glycoconjugate may comprise:
  • a probe as described herein may be provided by:
  • the step of creating group Y may include the step of reacting the monosaccharide units with a spacer moiety (Z).
  • Step (i) may involves forming an ⁇ -2,3 glycosidic bond between sialic acid and one of the one or more monosaccharide units.
  • Step (i) may involves forming an ⁇ -2,6 glycosidic bond between sialic acid and one of the one or more monosaccharide units.
  • the step of attaching three or more glycoconjugate ligands to a core moiety may include carrying out a 1,3 dipolar cycloaddition.
  • the 1,3 dipolar cycloaddition may involve cycloaddition of a propargyl-ether-moiety and an azide moiety.
  • Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent "about,” it will be understood that the particular value forms another embodiment.
  • Example 1 - alyconanoparticles presenting a trivalent ⁇ 2,6-thio-linked sialic acid can detect strains of human influenza within 30 mins in a simple colorimetric assay.
  • FIG. 1 A schematic representation of the aggregation of the glyconanoparticles in the presence of the influenza virus is shown schematically in Figure 1 .
  • the particles were used to detect the X31 virus from allantoic fluid (AF) at clinically relevant concentrations.
  • AF allantoic fluid
  • the ⁇ 2,6-binding trivalent sialic acid glyconanoparticles were shown to specifically detect human rather than avian influenza virus.
  • the synthesis of the monovalent ligand 3 is detailed in Scheme 2.
  • the aliphatic side chain of the alkyl thioglycoside of monovalent ligand 3 was synthesised starting from N- Boc-2,2'-(ethylenedioxy)bis(ethylamine) ( 11 ) prepared from the corresponding diamine following a published procedure. [25]
  • the mono- N -Boc-protected diamine 11 was then reacted with iodoacetic anhydride to give the corresponding iodoacetamide 12 in 70 % yield.
  • Compound 12 was then used for the formation of the thioglycoside 15.
  • the synthesis of monovalent ligand 3 started with the known ⁇ -thioacetate 13 [26,27] .
  • the UV-Vis spectrum in Figure 2a highlights the broadening of the surface plasmon absorption band of the 25:75 ratio trivalent ligand 1 :PEG gold nanoparticles indicating significant interaction with the influenza virus.
  • the same experiment was performed using gold nanoparticles functionalized with different ratios of monovalent ligand 3 :PEG ligand 2 (50:50, 25:75, 10:90, 5:95 and 2:98).
  • the results obtained suggest that a 25:75 ratio of the monovalent ligand 3 :PEG was also the optimum ligand density ( Figure 8 ).
  • the optimized glyconanoparticles were used to colorimetrically detect increasing concentrations of the X31 influenza virus.
  • Figure 2b upon addition of the human influenza virus X31 the surface plasmon absorption band red-shifted (from 525 to 536 nm) and decreased in intensity with increasing concentration of the virus ( Figure 2b and Figure 9a ).
  • the results suggest that the influenza virus induces aggregation of the glyconanoparticles as schematically shown in Figure 1 .
  • the aggregation of the optimized glyconanoparticles was spectroscopically measured 30 min following addition of increasing virus concentration. Changes of the surface plasmon absorption band due to the addition of the virus led to changes of solution color, from the initial deep red to lighter red ( Figure 9b ).
  • Trivalent ligand 1 was synthesised containing three ⁇ 2,6-thio-linked sialic acids. Human influenza virus binds preferentially to ⁇ 2,6 residues while avian influenza virus binds to ⁇ 2,3 residues. [14] Consequently, the optimized glyconanoparticles should not aggregate in the presence of avian influenza virus.
  • a thiolated trivalent ⁇ 2,6-thio-linked sialic acid derivative to functionalize gold nanoparticles.
  • the optimised glyconanoparticles consist of the thiolated trivalent ⁇ 2,6-thio-linked sialic acid derivative and a thiolated PEG derivative self-assembled onto the gold surface in a 25:75 ratio. These glyconanoparticles were used for the plasmonic detection of influenza virus.
  • the trivalent ligand 1:PEG (25:75) functionalized gold nanoparticles were used to detect the human influenza virus X31 (H3N2) within 30 min. Non-purified, influenza virus in allantoic fluid was successfully detected by the functionalized nanoparticles.
  • a comparison between the trivalent and a monovalent ⁇ 2,6-thio-linked sialic acid functionalized nanoparticles confirmed that more rapid results, with greater sensitivity, were achieved using the trivalent ligand for the detection of the X31 virus.
  • the trivalent ligand 1 :PEG (25:75) functionalized gold nanoparticles were able to discriminate between human ( ⁇ 2,6 binding) and avian ( ⁇ 2,3 binding) influenza. Since the dominant strain of human influenza varies seasonally, and with the possible threat of influenza virus crossing between animal species and thereby potentially initiating a pandemic, the ability to distinguish between human and avian influenza virus strains is exceptionally important.
  • the synthesis of a trivalent ⁇ 2,6-thio-linked sialic acid derivative to functionalize gold nanoparticles provides an innovative bioassay for the specific recognition and detection of influenza virus strains in clinical samples.
  • N -Boc aminopentanoic acid (valeric acid) was purchased from Sigma Aldrich. TLC was performed on precoated silica plates (Merck 60 F254, 0.25 mm) containing a fluorescence indicator.
  • NMR spectra were recorded on a Bruker spectrometer: 1 H NMR spectra recorded at 400 MHz were referenced to ⁇ H 7.26 for CDCl 3 or ⁇ H 3.34 for CD 3 OD; 13 C NMR spectra recorded at 100 MHz were referenced to ⁇ c 77.0 for CDCl 3 or ⁇ C 49.05 for CD 3 OD. Chemical shifts of NMR signals recorded in D 2 O are reported with respect to the methyl resonance of internal acetone at ⁇ H 2.22 ppm and ⁇ C 30.89 ppm, respectively. Assignments were made with the aid of COSY and HSQC experiments. Multiplicity of signals in 13 C NMR spectra was determined from HSQC spectra.
  • This oil was taken up in dry DMF (0.5 mL) and added to a mixture of commercial N -Boc aminopentanoic acid (86 mg, 0.4 mmol), HATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate) (0.2 g, 0.5 mmol), N -methylmorpholine (0.1 g, 1 mmol) in DMF (1 mL). The reaction was stirred at room temperature overnight, the DMF removed in vacuo, the resulting residue was taken up in CH 2 Cl 2 (25 mL), washed with water, dried (MgSO 4 ) and concentrated to dryness.
  • UV-Visible spectra were recorded using a Perkin Elmer Lambda 25 UV-Vis spectrometer at room temperature. Quartz cuvettes with a 1 cm path length were used. Transmission electron microscopy (TEM) images were obtained using a Jeol 2000EX transmission electron microscope, operating at 200 KV, by depositing samples on holey carbon film 300 mesh copper grids from Agar Scientific, UK.
  • TEM Transmission electron microscopy
  • Water soluble gold nanoparticles were prepared via the citrate reduction method reported by Enüstün and Turkevich. [5] Briefly, aqueous solutions of HAuCl 4 ⁇ 3H 2 O (12.5 mg, 32 ⁇ mol, in 100 mL) and sodium citrate tribasic dihydrate (50 mg, 168 ⁇ mol, in 50 mL) were prepared and heated to 60 °C. The sodium citrate solution was rapidly added to the gold solution while stirring vigorously. The temperature was increased to 85 °C and the solution was stirred for 2.5 h. A clear red gold nanoparticle solution was obtained that was cooled to room temperature and filtered through a Miller GP syringe driven filter unit (0.22 ⁇ m). The particle concentration in the citrate stabilized gold nanoparticles solution was approximately 3 nM.
  • Gold nanoparticles were functionalized with varying ratios of trivalent ligand 1 and PEG ligand 2. Varying molar ratios of trivalent ligand 1 and PEG based ligand 2 ( Table 4 ) were added to aliquots of freshly prepared gold nanoparticles (17 mL) and stirred for 60 h at room temperature to ensure self-assembly of the ligands onto the gold surface. The nanoparticle solution was centrifuged using Amicon Ultra-4 centrifugal filter units (10,000 MW cut-off) in a Sorvall Legend RT centrifuge for 10 min at 4,000xg to remove the excess trivalent ligand 1 and PEG ligand 2.
  • Gold nanoparticles were functionalized with varying ratios of monovalent ligand 3 and PEG ligand 2. Varying molar ratios of monovalent ligand 3 and PEG based ligand 2 ( Table 5 ) were added to aliquots of freshly prepared gold nanoparticles (17 mL) and stirred for 60 h at room temperature to ensure self-assembly of the ligands onto the gold surface. Excess ligands were removed as previously described for gold nanoparticles functionalized with trivalent ligand 1 and PEG ligand 2. Table 5. Molar ratios of monovalent ligand 3 and PEG ligand 2 added to the gold nanoparticles.
  • PEG ligand 2 (30.2 nmol) was added to a freshly prepared citrate stabilized gold nanoparticles solution (17 mL). The solution was stirred for 60 h at room temperature to ensure self-assembly of the ligand onto the gold surface. Excess ligands were removed as previously described for gold nanoparticles functionalized with trivalent ligand 1 and PEG ligand 2.
  • X31 virus (H3N2) (2.55 ⁇ g/mL) was added to a sample of each of the synthesized gold nanoparticles including: citrate coated gold nanoparticles; trivalent ligand 1 :PEG functionalized gold nanoparticles (50:50, 25:75, 10:90, 5:95 and 2:98); and monovalent ligand 3 :PEG functionalized gold nanoparticles (50:50, 25:75, 10:90, 5:95 and 2:98).
  • the samples were stirred at room temperature and the UV-Vis spectrum was recorded before addition of the virus and 0, 15, 30, 60 and 240 min after addition of the virus.
  • the UV-Vis spectrum of the sample was measured before addition of the AF X31 virus and 30 min after addition of the corresponding volume.
  • the same measurements were repeated although adding increasing volumes of Tris buffer (from 0 to 47.1 ⁇ L) to a sample of trivalent ligand 1 :PEG (25:75) functionalized gold nanoparticles (1000 ⁇ L).
  • avian RG14 virus H5N1
  • H5N1 avian RG14 virus
  • Each virus (6.8 ⁇ g/mL) was added to a sample of trivalent ligand 1 :PEG (25:75) functionalized gold nanoparticles.
  • the UV-Vis spectrum of each sample was measured after stirring the samples for 6 days at room temperature.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Nanotechnology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Claims (15)

  1. Sonde nanoparticulaire comprenant une pluralité de ligands glycoconjugués,
    chaque ligand glycoconjugué (GL) ayant une pluralité de groupes de reconnaissance contenant de l'acide sialique (Y) couplés à la nanoparticule via un coeur multivalent (X),
    dans laquelle le coeur multivalent (X) est un coeur trivalent, en conséquence de quoi il y a 3 groupes de reconnaissance par ligand,
    dans laquelle les groupes de reconnaissance sur le bioconjugué se lient spécifiquement à l'hémagglutinine sur le virus grippal cible,
    laquelle sonde a au moins un autre type de ligand lié à la nanoparticule, dans laquelle l'autre type de ligand est un polyéthylèneglycol (PEG) qui ne se lie pas spécifiquement à un virus grippal, dans laquelle chaque groupe de reconnaissance se termine par l'α-anomère d'un fragment d'acide sialique, et le fragment d'acide sialique est lié à un monosaccharide par l'intermédiaire soit :
    (i) d'une liaison 2,6-glycosidique, auquel cas le virus grippal cible est un virus grippal humain, lequel virus grippal humain est éventuellement un virus H3, soit
    (ii) d'une liaison 2,3-glycosidique, et le virus grippal cible est un virus grippal aviaire,
    et dans laquelle le rapport molaire du ligand glycoconjugué à l'autre ligand est compris entre 10/90 et 90/10.
  2. Sonde selon la revendication 1, dans laquelle le fragment acide sialique et le monosaccharide sont liés par liaison thio, et dans laquelle le monosaccharide est éventuellement le galactose.
  3. Sonde selon l'une quelconque des revendications 1 et 2, dans laquelle l'autre ligand est un PEG thiolaté.
  4. Sonde selon l'une quelconque des revendications 1 à 3, dans laquelle le rapport molaire du ligand glycoconjugué à l'autre ligand est compris entre 15/85 et 35/65, mieux encore entre 20/80 et 30/70.
  5. Sonde selon l'une quelconque des revendications 1 à 4, dans laquelle le rapport molaire du ligand glycoconjugué à l'autre ligand est d'environ 25/75.
  6. Sonde selon l'une quelconque des revendications 1 à 5, dans laquelle chaque sonde nanoparticulaire comprend un nombre moyen de molécules de ligand glycoconjugué égal à ou d'au moins 5, 10, 25, 50, 100 ou 200 par nanoparticule, et éventuellement la nanoparticule est une nanoparticule d'or, qui a éventuellement un diamètre de 10 à environ 30 nm.
  7. Sonde selon l'une quelconque des revendications 1 à 6, dans laquelle le GL répond à la formule :
    Figure imgb0103
     dans laquelle :
    Y est un groupe de reconnaissance contenant de l'acide sialique ;
    m = 3
    X est un fragment de coeur multivalent
    L est un fragment de liaison qui rattache le GL à la nanoparticule.
  8. Sonde selon la revendication 7, dans laquelle
    (i) L est une liaison thiol, et/ou
    (ii) X comprend un atome de carbone multivalent auquel trois groupes de reconnaissance Y sont liés via des groupes XL :
    Figure imgb0104
     où XL1, XL2 et XL3 sont choisis parmi -CH2-O-CH2 et -CH2-S-CH2- et où X est éventuellement un fragment de formule suivante :
    Figure imgb0105
     où f vaut 4 et g vaut 3,
    et où XL1, XL2 et XL3 sont -CH2-O-CH2-, et/ou
    (iii) Y comprend un fragment écarteur (Z) qui connecte le fragment contenant de l'acide sialique au groupe de coeur X,
    où Z comprend un groupe alkylène ou alcénylène qui contient éventuellement une ou plusieurs liaisons amine, amide, éther, ester ou thioester, et est éventuellement interrompu par un ou plusieurs hétéroatomes et/ou cycles aromatiques, et où Z est éventuellement un fragment de formule suivante :
    Figure imgb0106
     d vaut 3 et e vaut 5, et Y est éventuellement de formule suivante :
    Figure imgb0107
  9. Sonde selon l'une quelconque des revendications 1 à 8, dans laquelle le composé ligand glycoconjugué répond à la formule suivante :
    Figure imgb0108
  10. Procédé pour détecter spécifiquement un virus grippal cible dans un échantillon, lequel procédé comprend :
    (a) l'obtention d'une sonde nanoparticulaire selon l'une quelconque des revendications 1 à 9 ;
    (b) la mise en contact de la sonde nanoparticulaire et de l'échantillon dans des conditions efficaces pour lier spécifiquement l'hémagglutinine du virus grippal cible aux groupes de reconnaissance, dans laquelle ladite liaison spécifique génère un signal plasmonique détectable qui est spécifique du virus grippal ;
    (c) la détection du signal généré dans l'étape b).
  11. Procédé selon la revendication 10, dans lequel le signal détectable est un changement de couleur qui est observable à l'oeil nu, éventuellement dans lequel ladite liaison spécifique dans l'étape (b) provoque une agrégation d'une suspension aqueuse des nanoparticules, et dans lequel ladite agrégation génère ou contribue au signal plasmonique détectable.
  12. Trousse comprenant
    (i) une sonde nanoparticulaire selon l'une quelconque des revendications 1 à 9, ou
    (ii) une composition de détection de virus grippal comprenant une suspension aqueuse de sondes nanoparticulaires selon les revendications 1 à 9,
    dans chaque cas éventuellement avec des instructions d'utilisation pour la mise en oeuvre d'un procédé tel que défini dans la revendication 10 ou la revendication 11.
  13. Procédé pour préparer une sonde nanoparticulaire, lequel procédé comprend :
    (a) la préparation d'un composé ligand glycoconjugué tel que défini dans l'une quelconque des revendications 1 à 9 par :
    (i) création d'un groupe de reconnaissance contenant de l'acide sialique (Y) pour la liaison de HA par rattachement d'un ou plusieurs motifs monosaccharidiques à un acide sialique ;
    (ii) rattachement de trois groupes de reconnaissance contenant de l'acide sialique (Y) à un fragment de coeur (X) comprenant un fragment de liaison (L) ;
    (b) le rattachement du composé ligand glycoconjugué à une nanoparticule, via un fragment lieur (L).
  14. Procédé selon la revendication 13, dans lequel l'étape de création du groupe Y comprend l'étape de réaction des motifs monosaccharidiques avec un fragment écarteur (Z).
  15. Procédé selon la revendication 13 ou la revendication 14, dans lequel l'étape de rattachement de trois groupes de reconnaissance (Y) au fragment de coeur comprend la mise en oeuvre d'une cycloaddition 1,3-dipolaire, et dans lequel la cycloaddition 1,3-bipolaire met éventuellement en jeu la cycloaddition d'un fragment propargyl-éther et d'un fragment azoture.
EP14736939.1A 2013-07-24 2014-07-03 Détection de virus Active EP3024841B8 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB1313201.4A GB201313201D0 (en) 2013-07-24 2013-07-24 Virus Detection
PCT/GB2014/052026 WO2015011441A1 (fr) 2013-07-24 2014-07-03 Détection de virus

Publications (3)

Publication Number Publication Date
EP3024841A1 EP3024841A1 (fr) 2016-06-01
EP3024841B1 true EP3024841B1 (fr) 2018-11-21
EP3024841B8 EP3024841B8 (fr) 2019-02-27

Family

ID=49119197

Family Applications (1)

Application Number Title Priority Date Filing Date
EP14736939.1A Active EP3024841B8 (fr) 2013-07-24 2014-07-03 Détection de virus

Country Status (6)

Country Link
US (1) US10174069B2 (fr)
EP (1) EP3024841B8 (fr)
CN (1) CN105658657B (fr)
CA (1) CA2918674C (fr)
GB (1) GB201313201D0 (fr)
WO (1) WO2015011441A1 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160303242A1 (en) 2013-12-09 2016-10-20 Durect Corporation Pharmaceutically Active Agent Complexes, Polymer Complexes, and Compositions and Methods Involving the Same
MX2018005999A (es) * 2015-11-20 2018-11-22 Australian Biomedical Co Pty Ltd Compuestos para aplicaciones medicinales.
CN106589014B (zh) * 2016-12-05 2019-03-08 中国科学院微生物研究所 唾液酸寡糖-磁纳米粒子及其制备方法与应用
US11331019B2 (en) 2017-08-07 2022-05-17 The Research Foundation For The State University Of New York Nanoparticle sensor having a nanofibrous membrane scaffold
CN111698995A (zh) * 2018-02-17 2020-09-22 箭头药业股份有限公司 三炔连接剂和使用方法
US12065458B2 (en) 2018-02-17 2024-08-20 Arrowhead Pharmaceuticals, Inc. Trialkyne linking agents and methods of use
WO2022094932A1 (fr) * 2020-11-06 2022-05-12 深圳先进技术研究院 Bandelette réactive de détection de nouveau coronavirus, et son procédé de préparation et d'utilisation
WO2022094912A1 (fr) * 2020-11-06 2022-05-12 深圳先进技术研究院 Sonde biologique, son procédé de fabrication et son application
CN113984747B (zh) * 2021-10-21 2024-04-05 南京理工大学 一种金纳米阵列表面修饰唾液酸的方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080194801A1 (en) * 2007-02-14 2008-08-14 Swanson Basil I Robust multidentate ligands for diagnosis and anti-viral drugs for influenza and related viruses

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

Also Published As

Publication number Publication date
WO2015011441A1 (fr) 2015-01-29
EP3024841B8 (fr) 2019-02-27
US10174069B2 (en) 2019-01-08
EP3024841A1 (fr) 2016-06-01
CA2918674C (fr) 2021-06-15
CN105658657B (zh) 2020-03-03
GB201313201D0 (en) 2013-09-04
CA2918674A1 (fr) 2015-01-29
CN105658657A (zh) 2016-06-08
US20160185814A1 (en) 2016-06-30

Similar Documents

Publication Publication Date Title
EP3024841B1 (fr) Détection de virus
Hatano et al. Carbosilane glycodendrimers
He et al. Carbohydrate CuAAC click chemistry for therapy and diagnosis
KR101050774B1 (ko) 당 사슬-포착 분자로 당 사슬을 정제/농축하는 방법 및 당사슬 구조의 분석법
He et al. Fluorescent glycoprobes: a sweet addition for improved sensing
Kato et al. Development of tetraphenylethylene-based fluorescent oligosaccharide probes for detection of influenza virus
JP2005519627A (ja) バイオコンジュゲート‐ナノ粒子プローブ
Galante et al. Glycoclusters presenting lactose on calix [4] arene cores display trypanocidal activity
US20060057658A1 (en) Labeled substrate conjugates for identifying enzyme inhibitors
Oka et al. Syntheses and biological evaluations of carbosilane dendrimers uniformly functionalized with sialyl α (2→ 3) lactose moieties as inhibitors for human influenza viruses
JP2011153096A (ja) 蛍光性糖鎖プローブ
WO2008100553A1 (fr) Ligands multidentates robustes de diagnostics et médicaments antiviraux contre la grippe et des virus associés
Kumari et al. Synthetic assembly of novel avidin-biotin-GlcNAc (ABG) complex as an attractive bio-probe and its interaction with wheat germ agglutinin (WGA)
CN107917903A (zh) 糖基低维材料在甲型流感病毒荧光检测中的应用
CN115572335B (zh) 一种用于甲醛监测的壳聚糖基荧光探针及其制备方法和应用
CN108623711A (zh) 阿魏酸-环糊精共价偶联化合物及其制备方法和应用
US8513394B2 (en) Saccharide fluorescent substrates, preparation method and uses thereof
Montenegro et al. Multivalent assembly of a pyrene functionalized thio-N-acetylglucosamine: Synthesis, spectroscopic and WGA binding studies
CN110872335B (zh) 唾液酸寡糖-量子点缀合物、其制备方法及用途
CA2071915A1 (fr) Agent antiviral
CN102952207A (zh) 6-(1-甲基-β-咔啉-3-羧酸乙酰基)-6-脱氧-β-环糊精及其与阿霉素超分子包结配合物的制备和应用
KR100670948B1 (ko) 쿠커비투릴 유도체 단일 분자에 공유결합된 탄수화물다량체
keung Chan Synthesis And Functionality Study of Novel Biomimetic N-glycan Polymers
WO2005082918A1 (fr) Glycolipide en forme de bola polymérisable, agrégat tubulaire de celui-ci et polymère de celui-ci
JPH06247995A (ja) 新規なシアリル（α２−６）ラクトテトラオシルセラミド

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20160212

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20170207

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

INTG Intention to grant announced

Effective date: 20180524

GRAJ Information related to disapproval of communication of intention to grant by the applicant or resumption of examination proceedings by the epo deleted

Free format text: ORIGINAL CODE: EPIDOSDIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

GRAR Information related to intention to grant a patent recorded

Free format text: ORIGINAL CODE: EPIDOSNIGR71

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

INTC Intention to grant announced (deleted)
INTG Intention to grant announced

Effective date: 20181011

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

RAP2 Party data changed (patent owner data changed or rights of a patent transferred)

Owner name: ICENI DIAGNOSTICS LIMITED

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 1067389

Country of ref document: AT

Kind code of ref document: T

Effective date: 20181215

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602014036467

Country of ref document: DE

REG Reference to a national code

Ref country code: CH

Ref legal event code: PK

Free format text: BERICHTIGUNG B8

REG Reference to a national code

Ref country code: NL

Ref legal event code: MP

Effective date: 20181121

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 1067389

Country of ref document: AT

Kind code of ref document: T

Effective date: 20181121

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190221

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190321

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190221

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190222

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190321

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602014036467

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20190822

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20190731

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20190731

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20190731

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20190731

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20190703

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20140703

Ref country code: MT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181121

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IE

Payment date: 20230623

Year of fee payment: 10

Ref country code: FR

Payment date: 20230623

Year of fee payment: 10

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20230620

Year of fee payment: 10

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20230623

Year of fee payment: 10